The generation of neutrophils from hematopoietic precursors and their release to the peripheral circulation are highly regulated processes that ensure the maintenance of homeostatic neutrophil levels in the blood and their rise in response to bacterial infections and other signals. Altered neutrophil maturation and release are associated with various forms of neutropenia, which may precede and be pathogenetically linked to the development of myeloid leukemias. G-CSF has emerged a critical physiological regulator of granulopoiesis since mice carrying homozygous deletions of colony-stimulating factor (G-CSF) or its receptor are severely neutropenic, and dominant-negative mutations of G-CSFR have been linked to severe defects of granulopoiesis. Administration of G-CSF induces an expansion of myeloid lineage cells in the bone marrow, and promotes the release of neutrophils and hematopoietic progenitor cells from the bone marrow to the peripheral blood. Based on these properties, G-CSF is widely used to induce granulopoiesis and to mobilize hematopoietic progenitors to the peripheral blood. More recently, a CXCR4 competitive inhibitor, AMD3100/Plerixafluor, has been approved by FDA and a mobilizing agent for hematopoitic precursors in conjunction with G-CSF. Genetic studies and other studies have identified the transcription factor Gfi1 as a critical contributor to stem cells and myeloid cell function. Thus, mice null for Gfi1 fail to produce mature neutrophils, which has been attributed to a block in neutrophil maturation at the stage of common granulocyte/monocyte precursors. We have investigated the relationship between Gfi1 and G-CSF/G-CSFR in neutrophil maturation and their release from the bone marrow, and found that Gfi1 regulates G-CSF signaling. The biological activities of G-CSF are solely mediated by its activation of the G-CSF-receptor (R) that is expressed on myeloid lineage progenitor cells. Compelling evidence from genetic studies and other studies demonstrated that G-CSF indirectly promotes hematopoietic cell and neutrophil mobilization to the peripheral blood by modulating the activities of the chemokine SDF1 and/or its receptor CXCR4. WHIM, a genetic disorder associated with mutations in the intracellular domain of CXCR4 leading to increased CXCR4 function causes a retention of immature neutrophils into the bone marrow and severe peripheral neutropenia. AMD3100, a competitive inhibitor of SDF-1 binding to its receptor and a mutant form of SDF-1, which induces prolonged downregulation of the CXCR4 surface receptor, promote the mobilization of neutrophils and hematopoietic cells to the peripheral blood. During stem cell mobilization with G-CSF, SDF-1 and CXCR4 protein levels decrease in the bone marrow. We have examined the mechanisms responsible for reduced CXCR4 expression. Initially, we found that G-CSF reduces CXCR4 expression in bone marrow Gr1+ myeloid cells, which express G-CSFR. In additional studies, we have obtained evidence that the transcriptional repressor Gfi-1 is involved in G-CSF-induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We found that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF1 are reduced. Thus, Gfi1 not only regulates hematopoietic stem cell function and myeloid cell development but also likely promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function. In related experiments, we have generated mutants of CXCR4 that mimic mutations in the C-terminal domain found in patients with WHIM syndrome. We have examined the signaling mechanisms from wild-type CXCR4 and compared with signaling from mutants CXCR4 receptors. Our results indicate that unlike the normal receptor, mutant CXCR4 fails to appropriately recruit beta arrestin2, bur not beta arrestin1 to the receptor complex. As a consequence internalization of the mutant CXCR4 receptor from the cell surface to the cytoplasmic compartment is delayed, degradation is delayed, and signaling from the mutant receptor is also delayed. Since WHIM patients are heterozygotes for the mutant CXCR4 receptor and carry both the normal and the mutant allele, the net result is that CXCR4 signaling is extended in time, as it is the result of activation of both the normal and the mutant receptor. Thus, patients with WHIM have a super-functional CXCR4 receptor and presumably fail to release neutrophils from the bone marrow to the peripheral blood due to continuous signaling by the ligand SDF1, which holds the mature neutrophils in the bone marrow compartment. Since both the transcription factor Gfi1 and G-CSF/G-CSFR individually are critical contributors of myeloid cell differentiation from common myeloid/monocyte precursors in the bone marrow, we have investigated their relationship. We have uncovered a previously unrecognized function of Gfi1 as a regulator of G-CSF/G-CSFR signaling and function. Specifically, we found that Gfi1 regulates the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen-activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells partially rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. Mobilization of hematopoietic progenitor cells (HPC) from the bone marrow to the peripheral blood by G-CSF is the primary means to acquire stem cell grafts for hematopoietic cell transplantation avoiding invasive bone marrow collection. Since HPC represent a minority of all blood cells mobilized by G-CSF, there is a need for understanding the underlying mechanisms to develop selective drugs. We now found that G-CSF indirectly reduces expression of surface vascular cell adhesion molecule 1 (VCAM-1) on bone marrow HPC, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles/exosomes in the bone marrow extracellular compartment. We find that HPC, stromal cells and endothelial cells readily incorporate these exosomes, and that miR126 represses VCAM-1 expression on bone marrow HPC, stromal cells and endothelial cells. In line with this, miR126-null mice display a reduced mobilization response to G-CSF. Since mature neutrophils represent the main source of bone marrow microvesicles containing miR126, Gfi1-null mice that lack of mature neutrophils are defective in the mobilization of HPC. In addition, bone marrows of Gfi1-deficient mice have abnormally reduced levels of miR126, and express abnormally high levels of VCAM1 in the HPC. Altogether, our results implicate miR126 in the regulation of HPC trafficking between the bone marrow and peripheral sites, clarify the role of VCAM-1 in G-CSF-mediated mobilization, and have important implications for improved approaches to selective mobilization of HPC.